The murine cellular homolog of the 3611-murine sarcoma virus (MSV) v-raf oncogene was isolated as cDNA clones and characterized. Comparisons at the DNA and protein levels showed that the gene is under strict selection, as only 5 of the 95 nucleotide exchanges in the 3' half of the mouse and human c-raf genes resulted in amino acid differences. Twelve nucleotide exchanges occurred during the conversion of mouse c-raf to 3611- MSV v-raf. Eight of these were in the coding sequence and resulted in four amino acid exchanges. None of the differences coincide with those observed in the activation of v-mil, the avian homolog of raf, indicating that truncation and/or protein fusion, rather than point mutations, is the major factor in oncogene activation. The importance of conserved amino acid motifs for the kinase function of the raf protein was investigated by comparing the transforming potential of mutants generated by site-directed mutagenesis. The conversion of the second lysine in the putative ATP-binding site, Gly-X-Gly-X2-Gly-X1/3-Lys Ile - Leu-Lys, to either glutamine or glutamic acid did not significantly affect the transforming efficiency of the v-raf gene, while conversion of the first lysine to tryptophan eliminated the activity.